US7288246B2 - Method of alleviating chronic pain via peripheral glutaminase regulation - Google Patents

Method of alleviating chronic pain via peripheral glutaminase regulation Download PDF

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US7288246B2
US7288246B2 US10/245,098 US24509802A US7288246B2 US 7288246 B2 US7288246 B2 US 7288246B2 US 24509802 A US24509802 A US 24509802A US 7288246 B2 US7288246 B2 US 7288246B2
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glutaminase
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inhibitor
glutamate
pain
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Kenneth E. Miller
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University of Oklahoma
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect

Definitions

  • the present invention generally relates to methods of alleviating pain, and more particularly, but not by way of limitation, to a method of alleviating chronic pain by regulation of neurotransmitter synthesis.
  • Chronic inflammatory pain is a debilitating condition causing suffering, loss of work and loss of revenue.
  • Several methods of relieving pain from chronic inflammatory conditions such as rheumatoid arthritis, muscle damage, and osteoarthritis are known in the art.
  • the prior art methods of relieving pain have several unpleasant or serious side effects and require multiple daily administrations to be effective.
  • narcotics can be used for refractory chronic pain, but administration of narcotics has many side effects, including respiratory depression as well as the possibility of abuse.
  • another current method for relief of peripheral pain is topical application of capsaicin cream. This method may be effective for several days but produces severe acute pain in many patients.
  • some pain conditions such as myofascial pain and neuropathies due to nerve injury or disease currently do not have any effective therapies for alleviating pain associated therewith.
  • the present invention is related to a method of alleviating chronic pain in a subject for an extended period of time, as well as to a composition having analgesic effects that provides alleviation of chronic pain in a subject for an extended period of time.
  • the method of alleviating chronic pain of the present invention includes administration of an effective amount of at least one inhibitor of neurotransmitter synthesis into an inflammatory field.
  • Such inhibitor of neurotransmitter synthesis may be a glutaminase inhibitor.
  • One stimulator of sensory nerve fibers is glutamate produced by the sensory nerve fibers themselves.
  • Glutamate is a neurotransmitter utilized in signaling by the sensory neurons, and glutamate causes sensitization of surrounding sensory nerves, thereby producing the feeling of pain.
  • the present invention discloses that during experimental arthritis in rats, the sensory nerve cells increase production of glutaminase (GT), the neuronal enzyme that produces glutamate from glutamine.
  • GT glutaminase
  • Elevated amounts of glutaminase are shipped to the sensory nerve endings in the skin and joints, thereby causing increased amounts of glutamate to be produced (see FIG. 1 ).
  • the skin and joints from control rats have little to no detectable glutamate or glutaminase, so this enzyme and neurotransmitter have not been considered previously as possible therapeutic targets for pain relief via peripheral inhibition.
  • the method of the present invention includes local administration of an effective amount of at least one inhibitor of neurotransmitter synthesis, such as a glutaminase inhibitor, to a subject suffering from chronic pain at a site of inflammation, and the administration of the inhibitor of neurotransmitter synthesis results in a reduction in nociceptive responses, such as thermal and mechanical pain responses, at the site of inflammation for a period of at least two days without any resulting acute pain behavior.
  • at least one inhibitor of neurotransmitter synthesis such as a glutaminase inhibitor
  • rats were injected in the hindpaw with Complete Freund's adjuvant (heat killed Mycobacterium ) to create an experimental arthritis. Rats with this type of chronic inflammation have increased sensitivity to pressure and temperature. After several days of inflammation, some rats were injected with a glutaminase inhibitor, such as 6-diazo-5-oxo-L-norleucine (DON), N-ethylmaleimide (NEM), dicoumarol (DC), bromothymol blue (BB) and Palmitoyl Coenzyme A (P-CoA). Following application of the glutaminase inhibitor, the animal's sensitivities to pressure and temperature were brought to more normal values for many days, and these results were seen after only a single injection of the glutaminase inhibitor.
  • DON 6-diazo-5-oxo-L-norleucine
  • NEM N-ethylmaleimide
  • DC dicoumarol
  • BB bromothymol blue
  • the present invention also includes a method of alleviating both acute and chronic pain in a subject for an extended period of time.
  • the method includes administration of a combination therapy of an effective amount of at least one compound having analgesic effects that provides substantially immediate relief of acute pain in combination with an effective amount of at least one inhibitor of neurotransmitter synthesis to a subject suffering from acute and chronic pain at a site of inflammation.
  • Such combination therapy will provide relief of both acute and chronic pain and results in a substantially immediate reduction of nociceptive responses at the site of inflammation that last for a period of at least two days without any resulting acute behavior.
  • FIG. 1 is a diagrammatic representation of the effects of Glutamate and glutaminase on peripheral sensory nerve stimulation and exacerbation of pain responses.
  • FIG. 2 is a model regarding glutamate production in primary sensory neurons during chronic inflammation.
  • Inflammatory mediators (lightning bolts) activate and sensitize peripheral afferent terminals. This leads to the release of glutamate (GLU) and other substances from peripheral terminals causing further sensitization (arrow).
  • GLU glutamate
  • Inflammation stimulates keratinocytes to increase production of nerve growth factor (NGF).
  • NGF nerve growth factor
  • NGF nerve growth factor
  • GNF nerve growth factor
  • GNF nerve growth factor
  • GNF nerve growth factor
  • GNC glutaminase
  • Increased production of GT occurs from stabilization of GT mRNA via zeta-crystallin:quinone oxidoreductase (ZC).
  • ZC quinone oxidoreductase
  • Increased amounts of GT are shipped to the periphery causing elevated glutamate production and release, further primary afferent sensitization, and exacerbation of nocicept
  • FIG. 3 are photomicrographs illustrating the effects of fixation on glutaminase (GT) immunoreactivity (IR) in the rat dorsal root ganglia (DRG).
  • DRG sections were processed simultaneously with a mouse monoclonal GT antibody (A, C) or a rabbit polyclonal GT antiserum (B, D).
  • Some DRG's (A,B) were fixed with 4% paraformaldehyde and others (C,D) were fixed with 70% picric acid and 0.2% paraformaldehyde.
  • intense GT-IR was restricted to small sized DRG neurons (long arrows) with both GT antibodies (A,B).
  • FIG. 4 are photomicrographs illustrating Glutaminase (GT) immunoreactivity (IR) in rat L 4 dorsal root ganglia (DRG) following 7 days of CFA inflammation in the right hindpaw.
  • GT Glutaminase
  • IR immunoreactivity
  • DRG dorsal root ganglia
  • A In control sections, GT-IR was light to moderate in all neuronal cell sizes, small (long arrows) and medium to large (short arrows).
  • B Increased GT-IR intensity was observed in small (long arrows) and medium to large neurons (short arrows) in the left (contralateral) DRG following right hindpaw inflammation.
  • FIG. 5 is a graphic illustration of an image analysis of glutaminase (GT) immunoreactivity (IR) in L 4 DRG neurons after 7 days of CFA inflammation in the right paw. Data are presented as intensity divided by the area of the cell. DRG neurons were categorized into three area size groups: (A) small—100–600 ⁇ m 2 , (B) medium—600–1200 ⁇ m 2 , (C) large—>1200 ⁇ m 2 . (A) Small sized neurons in the left DRG contained a significantly greater immunoreactive signal (*, p ⁇ 0.05) than controls. Neurons in the right DRG were more intensely stained than left DRG or controls (**, p ⁇ 0.01).
  • GT glutaminase
  • IR immunoreactivity
  • FIG. 6 is a graphic illustration of GT enzyme activity in the L 4 DRG at 7 days following CFA inflammation in the right hindpaw.
  • GT activity from the right DRG (2.83 ⁇ 0.30 moles/kg/hr) was elevated (*, p ⁇ 0.05) over control values (2.20 ⁇ 0.18 moles/kg/hr).
  • the left (contralateral) L 4 DRG (2.61 ⁇ 0.20 moles/kg/hr) was not significantly different from controls or the right (ipsilateral) DRG.
  • FIG. 7 is a diagrammatic representation of the effects of inhibition of glutaminase on thermal and mechanical pain.
  • the hindpaw responses to thermal stimulation ( FIG. 7A ) and pressure sensitivity ( FIG. 7B ) were determined for a control rat, a control rat following glutaminase inhibition with 6-diazo-5-oxo-L-norleucine (DON), a rat after CFA inflammation, and a rat after CFA inflammation and following glutaminase inhibition with DON.
  • DON 6-diazo-5-oxo-L-norleucine
  • FIG. 8A is a graphic representation illustrating the efficacy of DON to provide long term pain relief from pressure (mechanical stimulation). After administration of DON at day three following CFA inflammation, pain relief occurred for several days with three different doses of DON (0.1–10 ⁇ Mole/25 ⁇ l).
  • FIG. 8B is a graphic representation representing the DON dose response for pain relief from pressure stimulation. The area under the curve for each dose was determined from Day 3 to Day 5. No differences in the amount of pain relief were determined for the doses tested (0.1–10 ⁇ Mole/25 ⁇ l).
  • FIG. 9A is a graphic representation illustrating the efficacy of DON to provide long term pain relief to heat. After administration of DON at day three following CFA inflammation, pain relief occurred for several days with three different doses of DON (0.1–10 ⁇ Mole/25 ⁇ l).
  • FIG. 9B is a graphic representation illustrating the DON dose response for pain relief from thermal stimulation. The area under the curve for each dose was determined from Day 3 to Day 5. Pain relief was most efficacious at the higher doses (1–10 ⁇ Mole/25 ⁇ l).
  • FIG. 10 are graphic representations illustrating that intraplantar injection of DON into the hindpaw of normal rats does not affect pressure or thermal senstivities.
  • DON was injected (10 ⁇ Mole/25 ⁇ l) on day three. Both the pressure ( FIG. 10A ) and thermal ( FIG. 10B ) sensitivities in DON-treated rats were the same as saline controls.
  • FIG. 11A is a graphic representation demonstrating the efficacy of N-ethylmaleimide (NEM) to provide long term pain relief to pressure (mechanical stimulation). After administration of NEM (10 mM/25 ⁇ l) at day three following CFA inflammation, pain relief occurred for several days.
  • NEM N-ethylmaleimide
  • FIG. 11B is a graphic representation illustrating the efficacy of NEM to provide long term pain relief from heat. After administration of NEM (10 mM/25 ⁇ l) at day three following CFA inflammation, pain relief occurred to near normal levels at days 4 and 6.
  • FIG. 12 are photomicrographs illustrating glutamate immunoreactivity in tissue sections from the hindpaw skin of a control rat ( FIG. 12A ), a rat after CFA inflammation ( FIG. 12B ), and a rat after CFA inflammation and following glutaminase inhibition with NEM ( FIG. 12C ).
  • FIG. 13A is a graphic representation demonstrating the use of two inhibitors at regulatory sites on glutaminase and their efficacy to provide long term pain relief to pressure (mechanical stimulation). After administration of Palmitoyl Coenzyme A (P-CoA, 2 mM/25 ⁇ l) or bromothymol blue (BB, 200 ⁇ M/25 ⁇ l) at day three following CFA inflammation, pain relief occurred for several days.
  • P-CoA Palmitoyl Coenzyme A
  • BB bromothymol blue
  • FIG. 13B is a graphic representation illustrating the efficacy of P-CoA and BB to give long term pain relief to heat.
  • FIG. 14 are photomicrographs illustrating that glutaminase production in many cells is regulated by zeta-crystallin:quinone oxidoreductase (ZC).
  • FIGS. 14A–C illustrate that ZC levels are modified during chronic inflammation.
  • ZC-immunoreactivity (IR) was examined in the rat L 4 DRG during inflammation at an early and later time point (2, 6 days).
  • ZC-IR in DRG neurons of control rats (A) shows a moderate staining of the cytoplasm of all neurons. Following inflammation for 48 hrs, ZC-IR is elevated in the cytoplasm and now appears in the nuclei of many neurons (arrows).
  • ZC-IR remains elevated at 6 days of inflammation and occurs mainly in the cytoplasm although some nuclei (arrows) contain light ZC-IR.
  • FIG. 15 is a diagrammatic representation that illustrates that dicoumarol, a ZC inhibitor, disrupts increased glutaminase production during chronic inflammation and decreases the prolonged hyperalgesia of chronic inflammation.
  • Inflammation was initiated with complete Freund's adjuvant (CFA) at Day 0, and dicoumarol (15 ⁇ l @ 500 ⁇ M) or saline was administered intrathecally on days 0, 1 and 2.
  • Thermal latencies and pressure responses (not shown) were recorded, and both the groups with inflammation (CFA) and inflammation plus dicoumarol (CFA+DC) experienced hyperalgesia and allodynia during acute inflammation (Day 1).
  • CFA+DC inflammation plus dicoumarol
  • the responses of CFA+DC rats became less hyperalgesic and allodynic.
  • the DRG's from the rats were collected and processed for glutaminase and ZC-IR, as shown in FIG. 16 .
  • FIG. 16 are photomicrographs illustrating that dicoumarol inhibits ZC and glutaminase production.
  • ZC-IR was elevated (A) in rats with inflammation, but the ZC-IR (B) from rats treated with DC during inflammation was similar to controls.
  • ZC-IR was found in the cytoplasm and nuclei (arrows) from rats with inflammation, whereas in rats treated with DC during inflammation, the nuclei (arrows) were not stained and ZC-IR was found primarily in the cytoplasm.
  • glutaminase-IR was observed at moderate levels from controls (C), elevated following inflammation (D), and similar to controls in rats treated with DC during inflammation (E).
  • the method of the present invention includes administration of an effective amount of at least one inhibitor of neurotransmitter synthesis to a subject suffering from chronic pain at a site of inflammation.
  • the inhibitor of neurotransmitter synthesis is a glutaminase inhibitor.
  • glutaminase inhibitors or “GT inhibitors” as used herein will be understood to include inhibitors that affect the activity of the glutaminase enzyme, such as inhibitors that may affect binding of glutamine, glutamate or various cofactors to the enzyme. That is, a GT inhibitor may block binding of the substrate glutamine to glutaminase, inhibit release of the product glutamate from glutaminase, or block cofactor binding and therefore slow the catalytic rate of the enzyme.
  • GT inhibitors examples include nonspecific inhibitors such as amidotransferase inhibitors and long chain fatty acids.
  • Specific examples of inhibitors of glutaminase activity which may be utilized in the method of the present invention include 6-diazo-5-oxo-L-norleucine (DON), N-ethylmaleimide (NEM), ⁇ -chloromercuriphenylsulfonate (pCMPS), L-2-amino-4-oxo-5-chloropentoic acid, DON plus o-carbamoyl-L-serine, acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid], azaserine, palmitoyl coenzyme A (palmitoyl CoA), stearoyl coenzyme A (stearoyl CoA), bromothymol blue, and combinations or
  • glutaminase inhibitors or “GT inhibitors” will also be understood to include inhibitors of glutaminase production.
  • Inhibitors of glutaminase production also include inhibitors of transcription of the gene encoding glutaminase as well as inhibitors of regulatory proteins involved in transcription of the glutaminase gene.
  • Inhibitors of glutaminase production also include inhibitors of translation of the glutaminase mRNA or inhibitors of stabilization of the glutaminase mRNA as well as compounds which increase degradation of the glutaminase mRNA. For example, as shown in FIG.
  • nerve growth factor is produced by keratinocytes in response to inflammation and is taken up and retrogradely transported to the neuronal cell body where it stimulates increased production of GT.
  • increased production of GT also occurs from stabilization of GT mRNA via zeta-crystallin:quinone oxidoreductase (ZC) ( FIG. 2 ). Therefore, a compound capable of neutralizing or inhibiting ZC or NGF also falls within the scope of the terms “glutaminase inhibitor” or “GT inhibitor”.
  • DC dicoumarol
  • the terms “glutaminase inhibitor”, “inhibitor of glutaminase enzyme activity” and “inhibitor of glutaminase synthesis” can all be used interchangeably herein.
  • the method of alleviating chronic pain of the present invention results in pain relief (both thermal and mechanical) for several days by way of peripheral glutaminase inhibition without any resulting acute pain behavior, as observed by the prior art methods, such as application of capsaicin cream. While the initial experiments described herein have utilized injection of an inhibitor of neurotransmitter synthesis, the inhibitor of neurotransmitter synthesis should also be amenable to topical or oral application. For example, an oral glutaminase inhibitor given as a prodrug or with limited to substantially no penetration into the central nervous system would also be effective in producing widespread pain relief.
  • the method of alleviating chronic pain of the present invention is not limited to injection of an inhibitor of neurotransmitter synthesis but also includes other methods of application of such inhibitor(s), such as, but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, intramuscular and intravenous routes, including both local and systemic applications.
  • the formulations containing at least one inhibitor of neurotransmitter synthesis described herein may be designed to provide delayed or controlled release using formulation techniques which are well known in the art. Using such methods of delayed or controlled release would provide an even longer period of pain relief.
  • subject as used herein will be understood to include a mammal, that is, a member of the Mammalia class of higher vertebrates.
  • mammal as used herein includes, but is not limited to, a human.
  • method of alleviating pain will be understood to include a reduction, substantial elimination or substantial amelioration of the condition of pain, including nociceptive behavior in response to mechanical or thermal stimuli.
  • nociceptive responses will be understood to refer to responses that occur in reaction to pain, such as mechanical or thermal stimuli.
  • pain as used herein will be understood to refer to all types of pain, including acute pain and chronic pain.
  • chronic pain as used herein will be understood to include, but is not limited to, pain associated with rheumatoid arthritis or osteoarthritis, neuropathic pain, pain associated with muscle damage, myofascial pain, chronic lower back pain, pain resulting from burns, and the like.
  • the present invention also includes a method of alleviating both acute and chronic pain in a subject for an extended period of time.
  • the method includes administration of a combination therapy of an effective amount of at least one compound having analgesic effects that provides substantially immediate relief of acute pain in combination with an effective amount of at least one inhibitor of neurotransmitter synthesis to a subject suffering from acute and chronic pain at a site of inflammation.
  • Such combination therapy will provide relief of both acute and chronic pain and results in a substantially immediate reduction of nociceptive responses at the site of inflammation that last for a period of at least two days without any resulting acute behavior.
  • Compounds having analgesic effects that may be utilized in such a method are known to those of ordinary skill in the art and include, but are not limited to, benzocaine, lidocaine, novocaine, and the like.
  • compounds which function as glutamate inhibitors or inhibitors of glutamate binding to glutamate receptors on peripheral sensory nerves may also be utilized as the compound having analgesic effects in the above-described combination therapy.
  • Other compounds having analgesic effects that may be utilized in the method of the present invention include aspirin, acetaminophen, paracetamol, indomethacin, cholinergic analgesics, adrenergic agents, nonsteroidal anti-inflammatory drugs, and other like compounds known in the art.
  • Compounds having analgesic effects are widely known, and it is well within the skill of a person having ordinary skill in the art to determine an effective amount of the compound having analgesic effects that will result in a reduction of acute pain upon administration to a subject.
  • CFA complete Freund's adjuvant
  • Sensory neurons respond chronically to inflammation by increasing tachykinin (substance P [SP]) and calcitonin gene-related peptide (CGRP) expression and content in dorsal root ganglia (DRG) [Calza et al, 1998; Donaldson et al, 1992; Garrett et al, 1995; Hanesch et al, 1993; Hanesch et al, 1995; Noguchi et al, 1988; Smith et al, 1992] and enhanced immunoreactivity in the spinal dorsal horn [Marlier et al, 1991], skin and joints [Ahmed et al, 1995; Nahin and Byers, 1994].
  • SP tachykinin
  • CGRP calcitonin gene-related peptide
  • peptide-containing neurons also are glutamatergic [Battaglia and Rustioni, 1988; De Biasi and Rustioni, 1988; Miller et al., 1993; Miller et al., 2002], using glutaminase (GT) as the synthetic enzyme for neurotransmitter glutamate production.
  • GT glutaminase
  • glutamate is released from central primary afferent terminals following noxious stimulation [Skilling et al., 1988; Sorkin et al., 1992; Yang et al., 1996].
  • Acute glutamate release in the spinal cord, along with SP and CGRP, is responsible for sensitization of spinal neurons leading to persistent or chronic changes [Dickenson, 1995; Pockett, 1995; Urban et al., 1994].
  • glutamate-immunoreactive fibers in the spinal cord increase 30% at 4 hr and nearly 40% at 8 hr [Sluka et al, 1992].
  • GT-IR in the DRG was evaluated with 2 fixatives and 2 antibodies.
  • a 4% PFA fixative small (100–600 ⁇ m 2 ) neuronal cell bodies were labeled intensely with GT-IR ( FIG. 3A , 3 B).
  • the 70% PA, 0.2% PFA fixative the majority of DRG neuronal cell bodies were labeled with both GT antibodies ( FIG. 3C , 3 D).
  • the PA-PFA fixative was used for the remainder of the experiments described herein.
  • the seven day rat immunohistochemistry images were analyzed with the SCION image analysis program in order to quantify the GT-IR intensities of three different sizes of DRG cell bodies ( FIG. 5 ).
  • the small (100–600 ⁇ m 2 ) DRG cell bodies showed the greatest amount GT-IR/area and the largest differences in intensities among control, left, and right cell bodies of the three different DRG cell sizes.
  • the small DRG cell bodies had intensities of 484.6 ⁇ 2.0/ ⁇ m 2 for controls, 532.6 ⁇ 1.7/ ⁇ m 2 for the left DRG from CFA rats, and 585.6 ⁇ 7.7/ ⁇ m 2 for the right DRG from CFA rats ( FIG. 5A ).
  • the GT-IR intensities for the medium (600–1200 ⁇ m 2 ) DRG cell bodies were 469.3 ⁇ 4.9/ ⁇ m 2 for the control, 509.6 ⁇ 8.9/ ⁇ m 2 for the left DRG from CFA rats, and 556.9 ⁇ 7.7/ ⁇ m 2 for the right DRG from CFA rats ( FIG. 5B ).
  • the GT-IR intensities for the large (>1200 ⁇ m 2 ) DRG cell bodies were 431.6 ⁇ 12.2/ ⁇ m 2 for the control, 448.5 ⁇ 10.7/ ⁇ m 2 for the left DRG from CFA rats, and 491.0 ⁇ 5.8/ ⁇ m 2 for the right DRG from CFA rats ( FIG. 5C ).
  • Control DRG's contained GT enzyme activity of 2.20 ⁇ 0.18 moles/kg/hr, whereas left and right DRG's from CFA rats had GT enzyme activities of 2.61 ⁇ 0.20 moles/kg/hr and 2.83 ⁇ 0.30 moles/kg/hr, respectively.
  • GT inhibitors were examined for their ability to alleviate nociceptive responses to thermal and mechanical stimuli.
  • GT enzyme activity includes 6-diazo-5-oxo-L-norleucine (DON) and N-ethylmaleimide (NEM).
  • DON 6-diazo-5-oxo-L-norleucine
  • NEM N-ethylmaleimide
  • DON irreversibly binds to the glutamine binding site of GT (Shapiro et al., 1979), whereas NEM partially inhibits GT via interaction with the glutamate binding site (Kvamme & Olsen, 1979; Kvamme & Lenda, 1982).
  • Intraparenchymal or ICV injection of DON inhibits GT and causes a decrease in glutamate and GT for several days in rat brain until neurons synthesize new GT (Bradford et al., 1989; Kaneko et al., 1992; Conti & Minelli, 1994). Therefore, DON and NEM were administered peripherally during chronic inflammation to observe the effect of GT enzyme inhibition on nociceptive responses.
  • CFA complete Freund's adjuvant
  • DRG neurons increase glutaminase (GT) production for shipment to peripheral terminals causing elevated glutamate (GLU) levels in skin and joints. Increased glutamate release may be responsible for maintaining thermal hyperalgesia and/or mechanical allodynia.
  • GT inhibitors including 6-diazo-5-oxo-L-norleucine (DON) and N-ethylmaleimide (NEM), were examined following inflammation.
  • CFA 6-diazo-5-oxo-L-norleucine
  • NAM N-ethylmaleimide
  • the efficacy of DON to provide long term pain relief to pressure was determined by using three different doses of DON (0.1–10 ⁇ Mole/25 ⁇ l). After administration of DON at day three following CFA inflammation, pain relief occurred for several days with all three doses of DON.
  • a dose response curve was constructed, as shown in FIG. 8B .
  • the area under the curve for each dose was determined from Day 3 to Day 5. No differences in the amount of pain relief were determined for the doses tested (0.1–10 ⁇ Mole/25 ⁇ l).
  • the efficacy of DON to provide long term pain relief to heat was determined for the same three doses of DON (0.1–10 ⁇ Mole/25 ⁇ l). After administration of DON at day 3 after CFA inflammation, pain relief occurred for several days with all three doses of DON. 10 ⁇ Mole DON ( ⁇ line) was most efficacious), bringing thermal responses back to normal for two days. The other two doses (0.1 and 1 ⁇ Mole, ⁇ and ⁇ lines, respectively) provided pain relief to near normal levels for at least one day and then gave variable results for the next several days.
  • a dose response curve was constructed, as shown in FIG. 9B .
  • the area under the curve for each dose was determined from Day 3 to Day 5. Pain relief was most efficacious at the higher doses (1–10 ⁇ Mole/25 ⁇ l).
  • FIG. 10 illustrates DON controls.
  • DON was injected (10 ⁇ Mole/25 ⁇ l) on day 3, and such injection of DON does not affect thermal or pressure sensitivities.
  • Both the pressure ( FIG. 10A ) and thermal ( FIG. 10B ) sensitivities in DON treated rats were the same as saline controls.
  • NEM N-ethylmaleimide
  • the skin from the hindpaws were also processed for GLU and GT immunohistochemistry after 7 days ( FIG. 12 ).
  • Control rats had very little GLU- or GT-immunoreactive (IR) fibers in the paw skin.
  • Skin from CFA+saline rats contained many intense GLU-IR and GT-IR fibers.
  • Skin from CFA+DON or CFA+NEM rats had moderate numbers of GLU-IR and GT-IR fibers.
  • P-CoA and BB are inhibitors of GT at regulatory sites on the enzyme.
  • P-CoA (2 mM/25 ⁇ l) or BB (200 ⁇ M/25 ⁇ l) was administered at day three following CFA inflammation, and both were shown to be effective in providing long term pain relief to pressure (mechanical stimulation, as shown in FIG. 13A ) and heat (thermal stimulation, as shown in FIG. 13B ).
  • FIG. 13A P-CoA ( ⁇ line) provided pain relief from Days 4–7, whereas BB ( ⁇ line) gave pain relief on Day 5.
  • P-CoA provided pain relief to near normal levels from Days 4–7
  • BB provided pain relief from Days 5–7 and at near normal levels from Days 6 and 7.
  • FIG. 14 illustrates that glutaminase production in many cells is regulated by zeta-crystallin:quinone oxidoreductase (ZC).
  • ZC levels are modified during chronic inflammation.
  • ZC-immunoreactivity (IR) was examined in the rat L 4 DRG during inflammation at an early and later time point (2, 6 days).
  • ZC-IR in DRG neurons of control rats (A) shows a moderate staining of the cytoplasm of all neurons. Following inflammation for 48 hrs, ZC-IR is elevated in the cytoplasm and now appears in the nuclei of many neurons (arrows).
  • ZC-IR remains elevated at 6 days of inflammation and occurs mainly in the cytoplasm, although some nuclei (arrows) contain light ZC-IR.
  • dicoumarol a ZC inhibitor
  • ZC a ZC inhibitor
  • the DRG contains high levels of GT enzyme activity [Duce and Keen, 1983; Graham and Aprison, 1969; McDougal et al., 1981], but localization of GT to specific neuronal cell types has been controversial to those of ordinary skill in the art.
  • Incubation of rat DRG's in [ 3 H]glutamine (converted to [ 3 H]glutamate via GT) labels neurons of all cell sizes [Duce and Keen, 1983].
  • Small sized neurons are stained exclusively with rabbit polyclonal GT antisera in PFA fixed tissue [Battaglia and Rustioni, 1988; Cangro et al, 1984, 1985], whereas most DRG neurons are stained using a mouse monoclonal GT antibody in PA-PFA fixed tissue [Miller et al, 1992, 2002].
  • GT immunostaining was compared with the 2 different fixatives and antibodies. In side by side comparisons, the same pattern of GT immunostaining occurred for both GT antibodies depending on the fixative used.
  • PFA fixative small sized DRG neurons were GT immunoreactive, but with PA-PFA fixative, the majority of the DRG neurons had GT-IR. This pattern is more consistent with glutamate immunohistochemistry where most DRG neurons are immunoreactive [Battagli and Rustioni, 1988; Stoyanova et al., 1998; Wanaka et al., 1987]. These results indicate that GT is sensitive to aldehyde fixation for detection with immunohistochemistry.
  • the increases in GT in the DRG after inflammation with complete Freund's adjuvant described herein further illustrate how primary sensory neurons are altered during chronic inflammation. If inflammation continues past the acute stage, the primary sensory neuron is induced into an altered phenotype making it more responsive to stimuli or sensitization.
  • sensory neurons respond chronically by modifying neuropeptide, receptor, and ion channel production [Calzà et al., 1998; Donaldson et al., 1992; Garrett et al., 1995; Gould et al., 1998; Hanesch et al., 1993, 1995; Millan, 1999; Mulder et al., 1997, 1999; Nahin and Byers, 1994; Noguchi et al., 1988; Seybold et al., 1995; Smith et al., 1992; Tate et al, 1998; Zhang et al., 1998].
  • DRG neuronal cell bodies have an altered phenotype that maintains or exacerbates inflammatory sensitization [Donnerer et al., 1992; Hanesch et al., 1993; Nahin and Byers, 1994; Ahmed et al., 1995; Garrett et al., 1995] and since most DRG neurons are glutamatergic [Miller et al., 1993, 2002a], it was necessary to determine if long-term alterations occur in glutamate metabolism of primary sensory neurons in chronic inflammation. Indeed, it has been shown herein that long-term elevated GT levels occur in DRG neurons during chronic inflammation.
  • SP and CGRP are found along with glutamate in primary afferent terminals [Merighi et al, 1991], and the co-release of glutamate and these neuropeptides generate hypersensitivity of spinal neurons [Besson et al, 1999]. Therefore, an increase in the amount of GT during chronic inflammation may lead to increased production and release of glutamate along with substance P and CGRP. Increased production and release of these substances could sustain spinal hypersensitivity maintaining a state of chronic pain.
  • Glutamate release occurs from peripheral processes [Bledsoe et al., 1980; Jackson et al., 1993; Lawand et al., 2000; Weinreich and Hammerschlag, 1975], and peripheral nerve terminals in skin contain glutamate receptors [Carlton et al., 1995, 1998; Carlton and Coggeshall, 1999; Coggeshall and Carlton, 1998].
  • long-term changes due to inflammation include an increase in glutaminase in the rat DRG cell body.
  • This increase in glutaminase will lead to elevated production and release of glutamate at both the peripheral and central processes of primary afferents.
  • An increase in glutamate metabolism in primary sensory neurons may be partly responsible for heightened nociceptive sensitivity in tonic pain models.
  • Prevention of increased glutaminase production or inhibition of glutaminase enzyme activity therefore, may reduce or block some nociceptive responses in inflammatory models.
  • NGF neurotrophic factor
  • DRG neurons also are glutamatergic, but the influence of NGF on glutamate metabolism in chronic inflammation has not been investigated.
  • NGF influences GT expression in DRG neurons in utero and in oculo [McDougal et al., 1981; Miller et al., 1999], and preliminary data indicate that NGF influences GT expression in the DRG and peripheral primary afferents similar to inflammation [Miller et al, 2001]. Therefore, it is believed that by inhibiting NGF's role on modifying glutamate metabolism in DRG neurons during chronic inflammation, GT expression and therefore glutamate levels can be reduced, thereby reducing nociceptive responses.
  • the rate of GT transcription is unaffected by these conditions, but the level of total and translatable GT mRNA is increased by stabilization of GT mRNA [Tong et al., 1987; Curthoys and Watford, 1995; Curthoys and Gstraunthaler, 2001]. Stabilization occurs by the binding of a cytosolic protein to an eight-base AU sequence repeat within the 3′-nontranslated region of the GT mRNA [Hansen et al., 1996; Laterza et al., 1997; Laterza and Curthoys, 2000; Porter et al., 2002].
  • This stabilizing protein is zeta-crystallin:quinone oxidoreductase [ZC; Tang and Curthoys, 2001; Curthoys and Gstraunthaler, 2001]. Since nervous system GT is similar or identical to kidney GT [Curthoys and Watford, 1995; Holcomb et al., 2000], it is possible that a similar mechanism exists in primary sensory neurons. Therefore, it is important to determine the role ZC has in increased GT production in DRG neurons during chronic inflammation. Several studies have shown altered levels of ZC in diseased neurons, tumor cells, and other tissues undergoing cellular stress [Wang et al, 2000; Siegel and Ross, 2000; Schelonka et al., 2000; Wilson et al., 2001].
  • ZC levels increase in the DRG neuronal cell bodies during the early stages of inflammation, preceding increases in glutaminase. Inhibition of ZC, therefore, was carried out to determine if glutaminase levels and pain behaviors could be modified.
  • DC Dicoumarol
  • ZC ZC is inhibited by several classes of compounds [al-Hamidi et al., 1997; Rabbani and Duhaiman, 1998; Winski et al., 2001; Bazzi et al., 2002].
  • Dicoumarol [DC] is a potent, competitive inhibitor of ZC, binding to the pyridine nucleotide site [Hollander and Ernster, 1975; Hosada et al., 1974, Jaiswal, 2000] and has been used as the traditional inhibitor of ZC in many studies [Cross et al., 1999; Winski et al., 2001]. Therefore, DC was administered to DRG neuronal cell bodies during chronic inflammation to disrupt ZC's regulation of GT production. The administration of DC caused a decrease in ZC and GT levels, as well as reducing nociceptive responses such as thermal hyperalgesia and mechanical allodynia.
  • Cutaneous primary afferents are classified into three general categories and proportions: 1. small diameter, unmyelinated, slow conducting C fibers [70%]; 2. medium diameter, lightly myelinated, intermediate conducting Adelta fibers [ 10 %]; 3. large diameter, myelinated, fast conducting Ab fibers [20%] [Millan, 1999]. Under normal conditions, nociceptors are categorized into Adelta fibers that evoke a rapid, acute pain sensation and C fibers that produce a later, ‘dull’ pain [Campbell, 1987].
  • Sensitizing substances released during acute inflammation include: 5-HT, histamine—mast cells; prosta-glandins (PG)—fibroblasts, Schwann cells; cytokines, H + , nitric oxide (NO)—macrophages; ATP, H + —damaged cells; 5-HT—platelets; ATP, NO—blood vessels; bradykinin, other kinins—blood; PG, neuropeptide Y, ATP—sympathetic terminals. There also is a neurogenic component of inflammation due to the release of bioactive substances from peripheral primary afferent terminals.
  • Substance P SP
  • CGRP calcitonin gene-related peptide
  • SP stimulated terminals or via axon reflexes (collateral fibers) further sensitizing surrounding afferent terminals and tissues.
  • These algogenic substances influence primary afferents to increase Ca 2+ and Na + permeability, decrease K + permeability, increase intracellular Ca 2+ concentration, NO and PG production, and adenylate cyclase and phospholipase C activities [Millan, 1999].
  • the peripheral primary terminal therefore, is acutely sensitized producing primary hyperalgesia.
  • Glutamate also is involved in neurogenic inflammation. As stated earlier, a number of stimuli evoke glutamate release from nerve trunks, skin, joints, and dental pulp [Bledsoe, et al., 1980, 1989; Jackson et al., 1993; deGroot et al., 2000; Lawand et al., 2000].
  • Fibers of the Ab type also contain EAA receptors [Coggeshall & Carlton, 1997; Wood & Mederty, 1997] and may be involved in mechanical allodynia [Millan, 1999].
  • EAA receptors Coggeshall & Carlton, 1997; Wood & Mederty, 1997
  • mechanical allodynia Millan, 1999.
  • This acute alteration in glutamate concentrations in peripheral primary afferents is due to local regulation of GT activity and glutamate production.
  • the present invention shows that chronic alterations in glutamate concentrations, however, involves increased production of glutaminase in the neuronal cell bodies followed by increased amounts of glutaminase and glutamate in the peripheral nerve fibers.
  • glutamate metabolism is altered for weeks in rat primary sensory neurons during chronic inflammation.
  • Elevated levels of glutamate and glutaminase (GT) occur in the neuronal cell bodies of dorsal root ganglia (DRG) followed by increases in the peripheral afferents of skin and joints.
  • DDG dorsal root ganglia
  • Chronic increase in production and release of glutamate can stimulate glutamate receptors on sensory afferents to produce hyperalgesia and allodynia. Therefore, elevated peripheral levels of glutamate cause exaggerated nociceptive responses during chronic inflammation.
  • zeta-crystallin:quinone oxidoreductase (ZC) is a stabilizer of GT mRNA to increase GT levels.
  • NGF nerve growth factor
  • ZC is a stabilizer of GT mRNA, allowing increased GT translation during times of cellular stress.
  • An effective amount of a ZC inhibitor can be administered to the DRG to disrupt GT mRNA stabilization and reduce nociceptive responses during the development of chronic inflammation.
  • NGF glutamate metabolism in primary sensory neurons
  • NGF has been implicated in chronic alterations of DRG neurons.
  • Administration of NGF to naive rats and NGF neutralization in chronic inflammation should have a similar effect as a ZC inhibitor on nociceptive behavior and glutamate metabolism in primary sensory neurons.
  • the L 4 DRG was examined for the following reason.
  • the tibial nerve a branch of the sciatic nerve, innervates the majority of the plantar surface of the rat hindpaw [Swett and Woolf, 1985].
  • Approximately, 99% of the tibial DRG neuronal perikarya of rats are located in the L 4 -L 5 DRG's, and the L 4 DRG contains more than twice the number than L 5 [Swett et al, 1991].
  • the threshold force was defined as the filament (force) that caused the foot retraction without bending the monofilament three out of five times. Using a conversion table for the filaments, thresholds were reported as gram force.
  • Thermal latencies for the footpad plantar surface were determined with the Hargreaves' model (Ugo Basile, Italy). Rats were placed on an elevated glass plate (3 mm) in clear plastic boxes with air holes in the lids and allowed to acclimate for 10 minutes. Radiant heat was applied to the plantar surface of the hindpaw and the withdrawal latency recorded. A second test was followed after 5–10 minutes. All behavioral testing occurred at 21–22° C. with indirect lighting in the testing room. Differences between groups for pressure thresholds and thermal latencies were determined with a Student's t test (p ⁇ 0.05 for significance) using InStat biological statistics program (GraphPad Software, Inc., San Diego).
  • PBS phosphate buffered saline
  • DRG sections from the first set of control rats were examined. Sections were incubated in rabbit anti-glutaminase (1:1000; gift from Dr. N. Curthoys, Colorado St. Univ., Ft. Collins, Colo.), mouse anti-glutaminase (IgM MAb 120, 1:500-5 mg/ml; gift from Dr. T. Kaneko, Kyoto Univ., Kyoto, Japan), or mouse anti-glutamate (1:3000; gift rom Dr. J. Madl, Colo. St. Univ., Ft. Collins, Colo.) in PBS-T.
  • tissue was washed three times in PBS and incubated in biotinylated goat anti-rabbit IgG or biotinylated goat anti-mouse IgM secondary antibody (5 ⁇ g/ml; Vector) in PBS-T for 1 hr.
  • Some tissue sections were washed two times in PBS following secondary antibody incubation, washed in sodium carbonate buffered saline (SCBS), pH 8.5, incubated in fluorescein-avidin (1.5 mg/ml; Vector) in SCBS for 1 hr, and washed three times in PBS.
  • Coverslips were apposed with Vectashield mounting media (Vector) to retard fading of immunofluorescence.
  • Sections were washed three times in PBS following secondary antibody, incubated in avidin-biotin-peroxidase (Vector), and washed three times in Tris-buffered saline, pH 7.6. Sections were incubated in diaminobenzidine (DAB) solution (0.5 mg/ml DAB, 0.003% H 2 O 2 in Tris-saline) for 1–5 minutes. Sections were dehydrated in an ascending series of ethanols, cleared in xylenes, and coverslips were apposed with Pro-Texx (Baxter Diagnostics).
  • DAB diaminobenzidine
  • a series of dilutions (1:200–1:6000) of the rabbit anti-glutaminase antiserum was used to determine an optimal dilution (1:3000) for evaluating alterations in immunohistochemical staining intensity.
  • a series of dilutions of the biotinylated goat anti-rabbit IgG secondary antiserum (1–15 ⁇ g/ml) was used to determine an optimal dilution (3 ⁇ g/ml) for this study.
  • Tissue sections for the CFA inflammation study were incubated overnight at 4° C. in rabbit anti-glutaminase (1:3000) in PBS-T and processed for immunofluorescence as described above. Immunofluorescent and immunoperoxidase sections were observed with an Olympus Provis AX70 microscope and digital images were obtained with a SPOTTM CCD camera (Diagnostic Instruments).
  • DRG's were evaluated qualitatively for 3, 7 and 10 day groups, and the 7 day group was chosen for quantitative densitometric analysis.
  • Immunofluorescent images from 7 day DRG's were captured using the CCD camera and saved as uncompressed TIFF files. Exposures were adjusted and pre-set by using experimental (CFA) images for baseline exposure.
  • CFA experimental
  • the glutaminase-immunoreactive DRG images were analyzed using the SCION Image program (Scion Co., Frederick, Md.). Individual DRG neurons were circumscribed, and the area, pixel number, and intensity were recorded. The data were recorded as intensity divided by the area of the cell.
  • Neuronal cell bodies in the DRG were distributed into the following three sizes for analysis: 100–600 ⁇ m 2 (small), 600–1200 ⁇ m 2 (medium), and >1200 ⁇ m 2 (large) [Willis and Coggeshall, 1991]. Differences in the intensity per area were analyzed with ANOVA followed by a Student-Newman-Keuls post hoc test (p ⁇ 0.05 for significance) using InStat biological statistics program (GraphPad Software, Inc.).
  • Enzyme assays for GT were performed according to the method of Curthoys and Lowry (1973). Five to six randomly selected sections of right and left DRG from rats with CFA and from control rats were placed individually in a 40 ⁇ l volume of reaction mixture containing: 20 mM glutamine, 100 mM K 2 HPO 4 , 0.6 mM EDTA, 0.01% Triton-X 100, 0.01% BSA in 50 mM TRIS, pH 8.65, for 45 minutes at 37° C. The reaction was stopped by adding 20 ⁇ l of 0.7 N HCl and placing the samples at 4° C.
  • a volume of 1 ml of indicator buffer containing 300 ⁇ M ADP, 360 ⁇ M NAD, 50 ⁇ g/ml glutamate dehydrogenase (GDH, rat liver, Boehringer Mannheim, Indianapolis, Ind.) in 50 mM TRIS, pH 8.5 was added for 20 minutes at room temperature.
  • glutamate produced by GT is converted to 2-oxoglutarate via GDH with the formation of NADH.
  • Reduction of NAD + was measured using a fluorometer (Farrand Inc.) with an excitation wavelength of 365 nm and emission at 340 nm. Quantitation of NADH production was accomplished by reacting multiple concentrations of glutamate standards in the indication reaction.
  • the GT activity from each DRG section was ascertained and a mean activity for each DRG was determined. Differences in GT activity from the left and right L 4 DRG's of CFA rats and L 4 DRG's from control rats were analyzed with ANOVA followed by a Student-Newman-Keuls post hoc test (p ⁇ 0.05 for significance) using InStat biological statistics program (GraphPad Software, Inc.).
  • Glutaminase immunoreactive neurons in the rat dorsal root ganglion contain calcitonin gene-related peptide (CGRP). Neurosci. Lett. (1993) 160:113–116.
  • CGRP calcitonin gene-related peptide
  • Islet amyloid polypeptide and calcitonin gene-related peptide expression are upregulated in lumbar dorsal root ganglia after unilateral adjuvant-induced inflammation in the rat paw. Brain Res. Mol. Brain Res. (1997) 50:127–135.
  • AMPA/kainate antagonist LY293558 reduces capsaicin-evoked hyperalgesia but not pain in normal skin in humans. Anesthesiology (1998) 89:1060–1067.
  • NAD(P)H quinone oxido-reductase 1 (NQO1) in human tissues. Free Radic Biol Med 29:246–253.

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